The present invention relates to magnetic particles exhibiting high sensitivity and low noise when a probe bonded with a biotin is bonded thereto.
Magnetic particles are used as a reaction solid phase of a diagnostic agent using an antigen-antibody reaction in order to detect substances to be examined such as infections, cancer markers, and hormones. In such a diagnostic agent, a probe (primary probe) for inspecting an antibody or an antigen is immobilized on particles. A substance to be inspected in a sample reacts with a second inspection probe after having been caught by the particles via the primary probe. The second inspection probe (secondary probe) is labeled with a fluorescent substance or an enzyme, whereby the target substance is detected by fluorescence or by an enzyme reaction. In recent years, due to a demand for an increase in the inspection sensitivity for early detection of diseases, an increase in sensitivity of a diagnostic agent has been an important subject. In order to increase sensitivity of diagnosis, a method of using enzyme coloring as a detecting means is being replaced by a method of using fluorescence or chemiluminescence, both of which ensure higher sensitivity.
Development of these detection techniques are said to have reached a level in which a one molecule-detection target can be theoretically detected. In practice, however, sensitivity is still insufficient. Generation of noise is given as the cause. Noise is generated by non-specific adsorption of secondary probes and impurities onto the surface of particles. For example, even if a technique that can theoretically detect a one molecule-detection target is used, detection of the one molecule is impossible if several molecules of a secondary probe are non-specifically adsorbed onto the surface of the particles. For this reason, a technique for lowering noise in order to reduce non-specific adsorption of a substance used for inspection onto the particle surface is strongly demanded.
Conventionally, a blocking method has been used for reducing such non-specific adsorption. In the blocking method, after immobilizing a primary probe on the particles, the particle surface is covered with a blocking agent such as albumin or skim milk with minimal adsorptivity of a secondary probe, impurities, and the like. However, some blocking agents may not exhibit a sufficient covering effect. Other blocking agents, which are biological substances, exhibit only poor quality stability. In these cases, even after complete blocking, the blocking effect may reduce over time and non-specific adsorption may occur due to denaturing of the blocking agent and the like. For these reasons, noise reduction by reducing non-specific adsorption has not been sufficiently attained.
In order to solve the problem of non-specific adsorption, a method of introducing a hydrophilic polymer onto the surface of a substrate for immunoassay represented by a 96-well plate has been proposed (JP-A-11-174057, JP-A-2000-304749, and JP-A-2001-272406). However, because the area available for immobilizing a primary probe is limited and the reaction of a primary probe with the target substance to be detected is a solid-liquid reaction, such an immunoassay substrate utilizing a flat surface has problems of poor efficiency of an antigen-antibody reaction, a long period of time required for inspection, and the like.
Furthermore, as countermeasures for decreasing non-specific adsorption, microspheres made from organic polymer particles of a styrene-glycidyl methacrylate copolymer and the like and a physiological active substance bonded to the organic polymer particles via a spacer (JP-A-10-195099, JP-A-2000-300283, WO 04/025297), organic polymer particles with a hydrophilic spacer introduced onto the particle surface (JP-A-2004-331953, WO 04/040305), and the like have been proposed. These organic polymer particles, however, exhibited neither a sufficient effect of reducing noise by reduction of non-specific adsorption nor sufficient immunoassay sensitivity.
The inventors have proposed magnetic particles for immunoassay exhibiting almost no non-specific adsorption, the particles having hydrophilic monomers such as a hydroxyalkyl(meth)acrylate, an alkoxyalkyl(meth)acrylate, a polyoxyalkylene (C2-C4) group-containing (meth)acrylate, an epoxy group-containing (meth)acrylate, and phosphorylcholine-analogous group-containing monomers copolymerized on the surface (JP-A-2005-69926). However, development of particles for immunoassay exhibiting higher sensitivity has been desired.